Accepted Manuscript, Basic and Clinical Neuroscience (Volume XX, Number XX) Full Title



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Accepted Manuscript, Basic and Clinical Neuroscience (Volume XX, Number XX)

Full Title: Effects of hydro-alcoholic extract of Anethum graveolens seed on pentylenetetrazol-induced seizure in adult male mice

Authors Name: 1- Mohammad Rostampour,

2-, Arghavan Ghaffari

3- Peyman Salehi

4- Farshid Saadat

Affiliations: 1- Cellular and Molecular Research Center, Guilan University of Medical Sciences, Rasht, Iran ; Department of Physiology, Guilan University of Medical Sciences, Rasht, Iran. Phone number: +98 131 6690099; Fax: +98 131 6690036; E-mail: rostampour@gums.ac.ir

2- Medical faculty of Guilan University of Medical Sciences, Rasht, Iran. Phone number: +98 131 6690099; Fax: +98 131 6690036 ; E-mail: Arghavan_g_67@yahoo.com

3- Shahid Beheshti University, Tehran, Iran. Phone number: +98 21 22431621; Fax: +98 21 22431586 ; E-mail: p-salehi@sbu.ac.ir

4- Department of Immunology, Guilan University of Medical Sciences, Rasht, Iran.; Cellular and Molecular Research Center, Guilan University of Medical Sciences, Rasht, Iran. Phone number: +98 131 6690099; Fax: +98 131 6690036 ; E-mail: fsaadat@razi.tums.ac.ir

Number of pages: Twenty pages (20)

Corresponding Author: Department of Physiology, Guilan University of Medical Sciences, Rasht, Iran. Phone number: +98 131 6690099; Fax: +98 131 6690036 ; E-mail: rostampour@gums.ac.ir

Running Title Anticonvulsant effect of Anethum graveolens seed hydro- alcoholic extract

Abstract

Introduction: Regarding to chronic nature of epilepsy, side effects and to access effective treatment procedures, herbal medicine has received remarkable interest. The aim of this study was to determine the anticonvulsant effects of hydro-alcoholic extract of Anethum graveolens seed on pentylenetetrazol (PTZ) -induced seizure in male mice.

Methods: Fifty-six albino male mice were divided randomly into seven groups including the negative control (saline), positive control (Phenobarbital) and treatment groups using different doses of hydro-alcoholic extract of Anethum graveolens seed (50, 100, 300, 500 and 1000 mg/kg). To provoke convulsion, PTZ was injected to all groups and initiation time of myoclonic and tonic-clonic seizures as well as surveillance after 24 h were measured.

Results: The results indicated that hydro-alcoholic extract of Anethum graveolens seed (AGS) delayed the initiation time of myoclonic and tonic-clonic seizures in comparison with saline group. The latency was considerable for myoclonic and tonic-clonic seizures at all above mentioned doses of AGS extract except for the lowest one. Moreover, the protective effect of AGS extract against mortality was statistically significant at all doses except for 50 mg/kg.

Conclusion: As the hydro-alcoholic extract of AGS showed an appropriate response in experimental model of convulsion, it might be considered as an adjuvant therapy with other traditional antiepileptic medications.

Keywords: Anethum graveolens, Pentylenetetrazol-induced seizure, mice

  1. Introduction

Epilepsy is a chronic neurological disorder of the brain that affects about 70 million people worldwide (Katchanov & Birbeck, 2012). It is characterized by recurrent involuntary movement accompanied by loss of consciousness which briefly termed seizures. The episodes are a consequence of excessive electrical discharges in a group of brain cells at different regions. Seizures can vary from the briefest lapses of attention, to severe and prolonged convulsions. Although, standard therapy permits control of seizures in about 80% of these patients, millions (500,000 people in the USA alone) have uncontrolled epilepsy (Roger, Porter, Brian, & Meldrum, 2009).

Regarding to chronic nature of epilepsy, resistance to chemical drugs, and lack of access to more effective treatment procedures, herbal medicine have received remarkable interest. Anethum graveolens is an annual herb of umbelliferae family which has been used traditionally to treat convulsion, stomach cramps and insomnia (Zargari, 1991). It's antimicrobial, anti-hyperlipidemic and anti-pyretic effects have also been reported (Hajhashemin & Abbasi, 2008; Mansouri et al., 2012). Additionally, the spasmolytic effect of Anethum graveolens seed (AGS) on rat uterus and ileum has been described (Gharib Naseri, Mard, & Farbod, 2005). Recently, some constituents of Anethum graveolens seed such as d-carvon, d- limonene, α- and β- pinene, linalool, and myrecene have been analyzed (Singh, Maurya, De Lampasona, & Catalan, 2005; Taher, Ghannadi, & Karimiyan, 2007). Concerning to several properties of dill on human body, we studied the effects of hydro- alcoholic extract of Anethum graveolens seed on PTZ – induced seizure in male adult mice.



2. Materials and methods

2.1. Chemicals and plant extraction

Pentylenetetrazol (PTZ) and Phenobarbital were purchased from Sigma-Aldrich (St. Louis, MO, USA). The seed of Anethum graveolens purchased from a local medicinal plants market and were identified by Dr. Ali Sonboli. A voucher specimen was deposited in the herbarium of Medicinal plants and Drugs Research institute, Shahid Beheshti University, Tehran, Iran (no. MPH-1966). . After cleaning and drying the plant seeds in room temperature, the seed powder (100 g) was macerated in ethanol 80% for 24 h at dark. Subsequently, the mixture was filtered and concentrated under reduced pressure at 40 ºC by rotary evaporator. The yield of hydro-alcoholic extract was 6.4% (g/g).



2.2. Animals

A total fifty-six albino male mice weighing 20-25 g were obtained from the animal house of School of Medicine, Guilan University of Medical Sciences. The animals were housed in standard cages with free access to food and water. The animal house temperature was 22 ± 2 ºc with a 12 hr light/dark cycle. The ethical guidelines for the investigation of experimental animals were followed in all tests in accordance with Guilan University of Medical Sciences ethical committee acts. Animals were randomly divided into seven groups (n=8). All groups were injected intraperitoneally (i.p). Negative control group received normal saline (10 ml/kg). Positive control group received Phenobarbital (40 mg/kg). Experimental groups received hydro- alcoholic extract of AGS at doses of 50, 100, 300, 500 and 1000 mg/kg. All groups were injected 45 min before administration of PTZ (Dhir & Kulkarni, 2006).



2.3. Pentylenetetrazol (PTZ) -induced seizure

In order to induce experimental model of epilepsy, PTZ (80 mg/kg) was dissolved in normal saline (0.9%) and injected intraperitoneally 45 min after administration of saline, phenobarbital and different amounts hydro-alcoholic extract of AGS (50, 100, 300, 500 and 1000 mg/kg). The animals were controlled after PTZ injection for 30 min. Then parameters of initiation time of myo-clonic and tonic-clonic seizures and percent of 24 h death were evaluated.



2.4. Data analysis

Data are expressed as mean ± SEM. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. In addition, Dunnett t-tests treat one group as a control, and compare all other groups against it. Protective effects of hydro-alcoholic extract of AGS against mortality after 24 h were evaluated by the Fisher’s Exact test. P < 0.05 was the critical criterion for statistical significance.



3. Results

3.1. Effect of hydro-alcoholic extract of AGS on latency of myo-clonic seizure

All animals in negative and treatment groups were shown seizure after PTZ administration. The results showed that all concentrations of extracts increased the initiation time of myo–clonic seizures in comparison with control group (normal saline). This escalation was significant at doses of 1000 and 500 mg/kg (P < 0.01) as well as 300 and 100 mg/kg (P < 0.05) (Fig.1).



3.2. Effect of hydro-alcoholic extract of AGS on latency of tonic-clonic seizure

As shown in Fig.2, all amounts of extract in comparison with saline group increased the initiation time of tonic–clonic seizures. The increment in time was significant at doses of 1000, 500 and 300 mg/kg (P < 0.01) and 100 mg/kg (P < 0.05).



3.3. Protective effect of hydro-alcoholic extract of AGS against mortality after PTZ -induced seizure

Hydro-alcoholic extract of Anethum graveolens seed decreased the percent of 24 h death as compared to control group. The protective effect against mortality (convulsion survivors/animals tested) of hydro-alcoholic fraction was 75% at a doses of 300 - 1000 mg/kg, 62.5% at a dose of 100 mg/kg and 25% at a dose of 50 mg/kg. According to table 1, this reduction was significant at doses of 300, 500 and 1000 mg/kg (P < 0.01) as well as 100 mg/kg (P< 0.05). Phenobarbital completely inhibited PTZ-induced seizures and percent of survival of animals after 24 h was %100.

4. Discussion

In the present study, it was indicated that the hydro-alcoholic extract of Anethum graveolens seed is effective on decrement of PTZ -induced seizure. Our finding is in agreement with Akaberi et al., study, in which they showed that aqueous extract of Anethum graveolens leaves had antagonistic effects on seizure induced by PTZ in mice (Akaberi, Mohammad-Zadeh, Mirmoosavi, Tazari, & Abarashi, 2013). Hence, the results of this investigation may underlie its traditional use in the treatment of convulsive disorder (Zargari, 1991).

Components which influence on experimental model of tonic–clonic seizures are considered useful in controlling myo-clonic and absence seizures in humans (Nisar, Khan, Simjee, Gilani, & Obaidullah, 2008). In fact, substances that inhibit convulsions or increase the latency of PTZ-induced seizures are suggested as having anticonvulsant activity (Haruna, 2000). Therefore, demonstration of AGS activity on seizure models suggests that this herb possesses anticonvulsant potential.

Here, we used hydro-alcoholic extract of dill to obtain more amounts of active components compared to water extract in accordance with Jana et al. results (Jana & Shekhawat, 2010). To achieve enough time for peritoneal absorption, extract administrated 45 min before induction of chemical convulsions (Dhir & Kulkarni, 2006). According to our findings, Anethum graveolens seed antagonized PTZ effect in a dose response manner based on the increase of latency of myo-clonic and tonic-clonic seizures in comparison with control group.

According to phytochemical screening depicted in Fig.3, various components such as monoterpenes have been isolated from Anethum graveolens (Taher, Ghannadi & Karimiyan, 2007). Monoterpenes such as carvone and limonene have protective effect against PTZ-induced convulsion (Sayyah, Moaied, & Kamalinejad, 2005; De Sousa, De Faras Nobrega, & De Almedia, 2007). Flavonoids and their derivatives have been found in AGS (Gebhardt et al., 2005; Moehle, Heller, & Wellmann, 1985). Flavonoids as an important class of natural compounds exert anti-oxidant properties (Dirscherl K et al., 2010). Since, chemical seizure induced by PTZ responds to antioxidant compound; flavonoids in AGS extract may be assumed potentially involved in anti-convulsant outcome (Pages et al., 2010).

Flavonoids have several neuropharmacological activities. Some of these effects are related to γ-aminobutyric acid type A (GABA)A receptors in the central nervous system (Abbasi, Nassiri-Asl, Shafeei, & Sheikhi, 2012). As, PTZ induces convulsion by antagonizing (GABA)A receptor chloride channel complex, manipulation of GABAA receptors affinity by flavonoids which in turn effect on CNS activity (Naseer, Shupeng, & Kim, 2009). Linalool as a further constituent of Anethum graveolens has an inhibitory effect on glutamate binding in the rat cerebral cortex (Elisabetsky, Marschner, & Souza, 1995). Since N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors are involved in epileptic activity, blockade of this receptor may also explain anticonvulsant effects of AGS extract (Da Silva, Witter, Boeijinga, & Lohman, 1990). Taken together, one of the probable explanations of dill anti-convulsant effect could be described by the existence of all above mentioned components in Anethum graveolens extract.

There is some evidence that Anethum graveolens show inhibitory effect on calcium L- type channels which are involved in convulsion (Gharib Naseri, Mard, & Farbod, 2005; Zamponi, Lory, & Prez-Reyes, 2010). This antagonistic effect may partially be explained by decrement in seizure initiation time obtained in this study.

In respect to the protective effect against mortality after PTZ -induced seizure, our findings indicate that all concentrations exert appropriate protection except for the attenuated one. Although, at least partly of these consequences is due to presence of the AGS components which induced suppression of nuclear transcriptional factor, other plausible mechanisms can not be anticipated at this step (Kim, Shin, Lee, & Kim, 2012).



5. Conclusion

Here, hydro-alcoholic extract of Anethum graveolens seed demonstrated anticonvulsant activity against PTZ-induced seizure. It might be considered as an adjuvant therapy with other traditional antiepileptic medications. Nevertheless, further studies are necessary for elucidation the involvement of probable neurotransmitter which mediated the functional mechanisms of whole extract.



Acknowledgments

The authors greatly appreciate Guilan University of Medical sciences for support and unit of medicinal plants of Shahid Beheshti University for preparing extract of plant.



Conflict of interest

There is no conflict of interest.



References

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Table legend

Table1. Effect of saline, phenobarbital and different doses of Anethum graveolens seed hydro-alcoholic extract on percent of 24 h death in eight rats in each group

Figure legends

Short title of Fig. 1: Latency of myo-clonic seizure

Fig. 1. The effect of different doses of Anethum graveolens seed hydro-alcoholic extract on latency of myo-clonic seizure when compared to control group (saline); Values are mean + SEM of eight mice in each group

* P < 0.05; ** P < 0.01 when compared to control group



Short title of Fig. 2: Latency of tonic-clonic seizure

Fig.2. The effect of different doses of Anethum graveolens seed hydro-alcoholic extract on latency of tonic-clonic seizure when compared to control group (saline); Values are mean + SEM of eight mice in each group

* P < 0.05; ** P < 0.01 when compared to control group


Short title of Fig. 3: Percent of constituents of AGS

Fig.3. The constituents of hydro-alcoholic extract of Anethum graveolens seed based on GC/MS analysis (Adopted from Taher et al. 2007)

Table1. Effect of saline, phenobarbital and different doses of hydro-alcoholic extract of Anethum graveolens seed on percent of 24 h death in eight rats in each group.

Groups Dose 24 h death %

Saline (negative control group) 10 (ml/kg) 100

Phenobarbital (positive control group) 40 (mg/kg) 0

Anethum graveolens seeds extract 1000 (mg/kg) 25**

Anethum graveolens seed extract 500 (mg/kg) 25**

Anethum graveolens seed extract 300 (mg/kg) 25**

Anethum graveolens seed extract 100 (mg/kg) 37.5*

Anethum graveolens seed extract 50 (mg/kg) 75


*P < 0.05; ** P < 0.01 when compared to control group



Fig.1. The effect of different doses of hydro-alcoholic extract of Anethum graveolens seed on latency of myo-clonic seizure when compared to control group (saline); Values are mean + SEM of eight mice in each group

* P < 0.05 ; ** P < 0.01 when compared to control group




Fig.2. The effect of different doses of hydro-alcoholic extract of Anethum graveolens seed on latency of tonic-clonic seizure when compared to control group (saline); Values are mean + SEM of eight mice in each group

* P < 0.05; ** P < 0.01 when compared to control group





Fig.3. The constituents of hydro-alcoholic extract of Anethum graveolens seed based on GC/MS analysis (Adopted from Taher et al. 2007)
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